Xylanases and cellulases are industrially important enzymes obtained from various microorganisms. Trichoderma harzianum is a preferred microorganism for the production of complete xylanases and cellulases. E-58 strain of the fungus was grown in different media including Enzyme Production Medium (EPM) and Vogel`s medium with different carbon sources like xylan, glucose and CMC to get the optimal production of xylanases and cellulases. It was found that glucose repressed the synthesis of these enzymes whereas xylan and CMC produced these enzymes in substantial amounts. Maximal activity of cellulases and xylanases was marked at pH 5.5 and at temperature 28°C. The fungus was grown for 5 days at 120 rpm in orbital shaker. Maximum enzyme activity of xylanase was found to be 1.209 IU mL-1 whereas maximum activity of cellulases (exoglucanase, endoglucanase and β-glucosidase) was found to be 2.764, 14.4 and 1.1209 IU mL-1 respectively.
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Cellulases and Xylanases are the industrially important enzymes. Celluloses, hemicelluloses and lignin are three major components of plant call wall. Cellulose is the most abundant organic polymer (Goyal et al., 1991) on the planet and is an important energy renewable source along with sugars and starches. Cellulase system contains three enzymes that can degrade crystalline cellulose i.e., Exoglucanase (E.C, 188.8.131.52), Endoglucanase (E.C, 184.108.40.206) and β-glucosidase (E.C.220.127.116.11). Xylan is major component of hemicellulose. It is also a major component of all monocots and hard woods, representing up to 35% of dry weight of these plants (Puls and Suchsil et al., 1993).
Xylanases have been extensively studied and could potentially be applied for the production of hydrolysates for agro-industrial wastes (Milagres et al., 1993), nutritional improvement of lignocellulosic foods, agro fibre (Bailey and Viikari, 1993), bioleaching of kraft pulps (Moreau et al., 1994) and in paper industry.
Cellulases are used in industries (Moo-Young 1985); in the preparation of medicines, resins, perfumes, in starch production, waste treatment, baking and in the production of plant protoplasts for genetic manipulation etc (Esterbauer et al., 1991).
Considering the industrial potentials of xylanases and cellulases, an important aspect of xylanase and cellulase research is to obtain highly active xylanases and cellulases at low cost.
The use of filamentous fungi may have a number of advantages over yeast and bacteria, the most important of which being their capability to be propagated on a wider variety of substrates generally discarded as wastes from food production and it involves simple harvesting operations (Sinskey, 1978). Some fungal species known for the production of xylanases and cellulases include Chaetomium thermophile, Trichoderma species and Aspergillus niger. Szczodrak (1988) found that Trichoderma reesei produces cellulases on wheat straw. Similarly different microbial sources have been investigated for β-xylanase production and strains of fungus Trichoderma harzianum have been shown to secrete large amounts of efficient xylan degrading enzymes (Wong and Saddler, 1992). The fungus Trichoderma harzianum was grown in Vogels medium, pH 5.5 and temperature 28°C at 120 rpm using xylan, CMC and glucose as carbon source. Earlier Rajoka et al. (1997) used cellulose, cellobiose, xylan and CMC for the enhanced production of cellulases. MacCabe et al. (1996) found that Xyln C production from T. reesei was induced by xylan.
In this paper we have reported the growth of T. harzianum on different media and the induction of xylanase and cellulase genes from the fungus by using different carbon sources.
MATERIALS AND METHODS
Selection of fungi: A fungal strain Trichoderma harzianum (E-58) strain was employed to check the induction of xylanase and cellulase genes using different carbon sources. The fungus was obtained from National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad.
Substrates: Different substrates (carboxymethycellulose (CMC), xylan and glucose) were used for the induction of the cellulose xylanase genes from Trichoderma harzianum.
Media and culture conditons
|A.||Trichoderma harzianum E-58 was maintained on agar slants (MYJ) containing (g L1) malt extract 5; yeast extract 2.5; glucose 10; agar 20. Freshly inoculated slants were incubated at 28°C for 5 days and stored at 4°C (Ulho and Peberdy, 1991).|
|B.||Production of Xylanases and cellulases was carried out in one-liter flasks containing 250 mL of EPM (Enzyme Production Medium). EPM comprises (g L1) glucose 3, bactopeptone 1; urea 0.3; (NH4)2SO4 1.4; MgSO4.7H2O 0.3; CaCl2.6H2O 0.3: Chitin 5.0; and 0.1% vol vol1 trace elements solution containing (Fe2+,Mn2+, Zn2+and Co2+).|
|C.||For the induction of xylanases and cellulases genes, the fungus was grown at 28°C with shaking at 120 rpm in Vogels medium (0.5% Trisodium citrate, 0.5% KH2PO4, 0.2% NH4NO3, 0.4% (NH4) 2 SO4, 0.02% MgSO4, 0.1% peptone, 0.2% yeast extract, pH 5.50) containing 1% glucose or CMC or xylan as a carbon source (Ikram-ul-Haq et al., 2000).|
Inoculum: Flasks were autoclaved at 121 °C for 15 minutes and inoculated with a loopful of fungal spores suspended in 100 mL of Vogels medium. The flasks were incubated at 28°C. The incubation time was limited to 24 h with shaking at 120 rpm in orbital shaker.
Xylanase assay: Activity of the enzyme was checked by Miller method (1959). One mL citrate phosphate buffer, 0.5 mL Xylan (1%) and 0.5 mL of diluted enzyme mixture were incubated at 50°C for 30 min. After expiry of time 3 mL DNS reagent was added and mixture was placed in boiling water bath for 5 min and the absorbance was noted at 550 nm. The international units of xylanase produced per mL were calculated by factor obtained from standard curve of xylose (Fig. 1).
Exoglucanase assay: Exoglucanase assay is based on the fact that the enzyme releases free cellobiose from avisel. One milliliter of diluted enzyme extract was incubated with 1 mL of 0.1 mL citrate buffer at pH 4.8 at 40oC for 30 min. The reaction was stopped by boiling for 15 minutes and cooling in ice. After cooling, absorbance was measured at 540 nm. The international units of exoglucanase produced mL1 were calculated by factor obtained from the standard curve of cellobiose (Fig. 2).
|Fig. 1:||Standard curve of xylose|
|Fig. 2:||Cellobiose standard curve|
|Fig. 3:||Standard curve of glucose|
|Fig. 4:||Standard curve of p-nitrophenol|
Endoglucanase assay: It was performed as described by Gadgil (1995). Briefly 1 mL of appropriately diluted enzyme (0.01mL) was incubated for 30 minutes with 1 mL of 1% CMC and 1 mL of 0.1M citrate buffer of pH 4.8 at 50oC. The reaction was trimmed by adding 3 mL of DNS reagent and boiled for 15 min and cooled in ice. The international units of endoglucanase produced mL1 were calculated by factor obtained from standard curve of glucose (Fig. 3).
β-glucosidase assay: It is based on the principal that p-nitrophenyl β-D-glucopyranoside is hydrolyzed by β-glucosidase to yield β-D-glucose and p-nitrophenol. One mL of β-D-glucopyranoside solution (substrate) in buffer and 1 mL of appropriately diluted enzyme were added and incubated at 40oC for 10 min. Three mL of 1M Na2CO3 (Stopper) were added and volume was made up to 10 mL with distilled water. Absorbance was measured at 400 nm. The international units of β-glucosidase produced mL1 were calculated by factor obtained from standard curve of p-nitrophenol (Fig. 4).
RESULTS AND DISCUSSION
Growth of Trichoderma harzianum:The fungus Trichoderma harzianum was employed in this research. Earlier Esterbauer et al. (1991) found that Trichoderma strains are preferred microorganisms for the production of complete cellulases. Wong and Saddler (1992) studied properties and applications of xylanases from Trichoderma spp, which produced these enzymes with high xylanolytic activity. They isolated and purified different xylanases form Trichoderma species.
EPM (enzyme production medium) was used for the growth of fungus but growth did not come out. However Ulhoa et al. (1991) obtained positive results from EPM for the growth of Trichoderma harzianum for large-scale production of chitobiase. MYG medium (Heesing et al., 1994) was tried for sporulation of the Trichoderma harzianum in agar slants but desirable results could not be achieved. After failing to get the growth of Trichoderma harzianum on EPM and MYG media, Vogels medium was used for the growth of Trichoderma harzianum. Spores of T.harzianum were found on agar slants containing 0.2% xylan or CMC. The spores were inoculated in shake culture. T. harzianum was grown on Vogels medium for 5 days at 120 rpm.
Optimum pH for Trichoderma harzianum: The pH of the Vogels medium was adjusted to 5.5 to obtain the maximal production of the xylanases and cellulases from Trichoderma harzianum. Earlier Simpson (1991) worked on the production of thermostable endo 1-4 β xylanase from culture supernatant of Thermogota spp and noted that the optimum pH was between 5.0 and 5.5 for its activity. Rose and Van (2002) found that xylanase enzyme expresses its highest activity at pH 5-6. Similarly De Paula-Silveira et al. (1999) found that xylanase from T. harzianum had a maximum activity at pH 5.0. Milagres et al. (1993) worked on the production and characterization of xylanase from Penicillum Janthium and found that optimal pH for xylanase activity was 5.5.
Srivastava et al. (1984) used cellobiose and 4 nitro phenyl β-D glucosidase as substrate for the growth of Aspergillus Wentii to study the kinetic characteristics of β-D glucosidase and glucohydrolase. They found that the enzyme activity was maximum over a pH range of 4.5 to 5.5 for both substrates. Similarly it was found earlier that maximum induction of endoglucanase was achieved at pH 5.5 (Ikram-ul-Haq et al., 2000).
Temperature and shaking: Temperature of the medium was kept at 28°C with shaking at 120 rpm, which is in accordance with the previous work conducted (Sexton et al., 2000). Esterbauer et al. (1991) found that optimum temperature for cellulase production form Trichoderma reesei was 15-28°C and optimal temperature for its growth was 30°C. Similarly Gomes et al. (1992) used Trichoderma viridie Bt 2169 for the production of cellulases and xylanases. They noted that the optimum temperature for the maximum production of these enzymes was 31.1oC. Smith and Wood (1991) observed that the optimum temperature was 30oC and 35oC for the production of extracellular xylanases and β xylosidase by Aspergillus awamori.
Induction of Xylanase and Cellulase genes from different substrates: Trichoderma harzianum was grown in Vogels medium at 28°C for 5 days at 120 rpm for the maximal production of cellulases and xylanases with different carbon sources like CMC, xylan and glucose.
|Table 1:||Xylanase and cellulase concentrations produced from T. harzianum|
After 5 days the culture mixture was taken out of orbital shaker. The enzyme filtrates obtained were tested for enzyme activities. Enzyme assays were performed as described in materials and methods to check the activity of xylanases and cellulases. The results obtained are summarized in Table 1. The results are in accordance with those of earlier work conducted on Trichoderma harzianum (De Paula-Silveira et al.,1999, Emilo et al., 1996).
Present results suggest that when glucose was used as a carbon source, the production of xylanases and cellulases was inhibited and when xylan or CMC was used as a carbon source the production increased significantly. The results are in agreement with the earlier reports where it was found that XylnC production from Aspergillus nidulans was induced by xylan and repressed by glucose (MacCabe et al. (1996)). Zeilinger et al. (1996) found that xyln2 production from T. Reesei was induced by the presence of xylan and xylose.The fungus Aureobasidium pullans Y2311-1 has been reported to produce highest level of xylanase when xylan was used as a carbon source (Liang and Ljundahl, 1994). Two of endo beta 1,4 xylanases were isolated from culture filtrates of T.reesei C30 grown on xylan as a carbon source (Torronen et al., 1992).
Earlier Rajoka and Malik (1997) used cellulase, cellulosic residues, xylan, cellobiose and CMC as a carbon source for the enhanced production of cellulases by Cellulomonas strain. Ximenes et al. (1996) found that high glucose repressed the induction of cellulases whereas low glucose potentiated the enzyme production. Previously it was found by Northern analysis that genes of cellobiohydrolases were induced with 1% cellulose or CMC.
The effect of carbon sources on T.harzianum was also studied by Malik and co workers (1986). They optimized the fermentation conditions and found that maximum growth was obtained with glucose as a carbon source but negligible cellulases were produced. They suggested that substantial amounts of cellulases were produced from other carbon sources. Esterbauer et al. (1991) used CMC and xylan as a carbon source for the maximal production of glucosidase.
We, therefore, conclude that the optimum conditions for the growth of T.harzianum were at pH 5.5, temperature 28oC and shaking at 120 rpm in Vogels medium. It was found that the xylanase and cellulase genes were induced when xylan or CMC were used as a carbon source and repressed by glucose.
The authors are especially thankful to Dr. M. I. Rajoka and Dr. Farooq Latif from Natinal Institute for Biotechnology and Genetic Engineering, Faisalabad for providing Trichoderma harzianum. The work was supported by grant from Pakistan Science Foundation and Ministry of Science and Technology, Government of Pakistan.
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